Fluidization of brain membranes by A2C does not produce anesthesia and does not augment muscimol-stimulated Cl influx

Intravenous administration of 2-[2-methoxyethoxy]-ethyl 8-[cis-2-n-octylcloropropyl]-octanoate (A₂C) was found to disorder brain membranes but did not produce intoxication or anesthesia in mice. The abilities of A₂C and an anesthetic (benzyl alcohol) to inhibit [³⁵S]t-butylbicyclophosphorothionate (TBPS) binding, and modify γ-aminobutyric acid (GABA) receptor-mediated ³⁶Cl⁻ influx into brain vesicles were then compared. Both of the perturbants inhibited [³⁵S]TBPS binding at the same concentrations at which they reduced membrane order; however, the anesthetic was nearly 4 times more effective in reducing [³⁵S]TBPS binding than was A₂C. Muscimol-stimulated ³⁶Cl⁻ uptake was enhanced by benzyl alcohol at a concentration which produced little or no change in membrane order. Concentrations of both A₂C and benzyl alcohol which reduced membrane order inhibited muscimol-stimulated ³⁶Cl⁻ influx. Similarly, membrane order and muscimol-activated ³⁶Cl⁻ uptake were reduced in brain vesicles prepared from mice which had received A₂C in vivo. The effects of anesthetics on the GABA_A receptor-chloride channel complex were analyzed by a two site model of action in which a ‘perturbant’ site is responsible for decreased ³⁶Cl⁻ uptake; but a distinct ‘anesthetic’ site is responsible for augmentation of chloride flux and anesthesia.     

Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain.

The inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA_A receptor by the insecticide γ-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC₅₀ from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC₅₀ values found in the literature. IC₅₀ values within the range of 150–250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [³⁵S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.     

Direct Activation of GABAA Receptors by Loreclezole, an Anticonvulsant Drug with Selectivity for the β-Subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates γ-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor β-subunits. A similar selectivity for GABA_A receptor β-subunits is apparent for the direct activation of receptor-operated Cl⁻ channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABA_A receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding by up to 28% (at 5 μM) and 80% (at 10 μM), respectively. Higher concentrations (50–100 μM) of both compounds inhibited [³⁵S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [³⁵S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50–100 μM, loreclezole induced inward Cl⁻ currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABA_A receptors, comprised of ₁-, β₂- and γ_2S-subunits. At 100 μM, the current evoked by loreclezole was 26% of that induced by 5 μM GABA. The current evoked by 100 μM propofol was 98% of that induced by 5 μM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABA_A receptors containing the β₂-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABA_A receptors containing the β₁-subunit may be responsible for its lack of hypnotic effect. © 1997 Published by Elsevier Science Ltd. All rights reserved.     

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the “cage” convulsant [³⁵S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [³⁵S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [³⁵S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.     

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Pyrethroid insecticides indirectly inhibit GABA-dependent Cl influx in synaptoneurosomes from the trout brain

Rainbow trout (Oncorhynchus mykiss) are extremely sensitive to the neurotoxic activity of pyrethroid insecticides. One possible target for pyrethroids is the GABA_A receptor of brain of the trout, the function of which can be tested by measurement of influx of ³⁶Cl ⁻ into synaptoneurosomes, in response to the application of agonists. γ-Aminobutyric acid produced a time- and concentration-dependent increase in influx of ³⁶Cl⁻ in synaptoneurosomes from the brain of the trout, which exhibited the pharmacology characteristic of a response mediated by activation of a GABA_A receptor. Deltamethrin, (1RS)-cia-cypermethrin and permethrin produced a dose-dependent increase in the basal uptake and a corresponding decrease in GABA-dependent influx, with a maximum inhibition of 70–82%. This effect of pyrethroid was stereospecific, of high potency and inhibited by tetrodotoxin (TTX) and t-butylbicy-clophosphorothionate (TBPS). The sensitivity of the effect of the pyrethroid to TTX suggested an activation by pyrethroid of the voltage-dependent sodium channel. Veratridine, a sodium channel activator, elicited similar changes in the basal uptake of chloride, which were TTX-sensitive. Neither deltamethrin nor veratridine had a measurable effect on the efflux of ³⁶Cl⁻ from synaptoneurosomes. Thus, pyrethroid insecticides may interfere with the function of GABA_A receptors indirectly through an interaction with the voltage-dependent sodium channel in the brain of the trout and consequently perturb chloride influx, possibly through a voltage-dependent chloride channel.     

Autoradiographic analysis of cannabinoid receptor binding and cannabinoid agonist-stimulated [S]GTPγS binding in morphine-dependent mice

The present study was designed to test the possible existence of changes in brain cannabinoid receptors in morphine-dependent mice. To this end, we compared cannabinoid receptor binding and WIN 55,212-2-stimulated [³⁵S]guanylyl-5′-O-(γ-thio)-triphosphate ([³⁵S]GTPγS) binding in several brain regions of mice chronically exposed to morphine or saline. The existence of opiate dependence in morphine-injected mice was assessed by analyzing the well-known jumping behavior induced by the blockade of opioid receptors with naloxone, whereas these animals were unresponsive to the blockade of cannabinoid receptors with SR141716. The different structures analyzed exhibited similar cannabinoid receptor binding levels in morphine-dependent and control mice, with the only exception of the globus pallidus, which exhibited a very small, but statistically significant, increase. In addition, the activation of cannabinoid receptors with WIN 55,212-2 increased [³⁵S]GTPγS binding in most of the structures examined. The increase was of similar magnitude in morphine-dependent and control mice, except in the substantia nigra, where morphine-dependent mice exhibited lesser [³⁵S]GTPγS binding levels in basal conditions, although a significantly higher WIN 55,212-2-stimulated binding. Other structures, such as the central gray substance, where there was a poor agonist-induced stimulation in control mice, exhibited, however, higher levels of WIN 55,212-2-stimulated [³⁵S]GTPγS binding in morphine-dependent mice, whereas these animals tended to exhibit a higher [³⁵S]GTPγS binding levels in basal conditions, although a lesser and not statistically significant WIN 55,212-2-stimulated binding, in the deep layers of the cerebral cortex. Thus, the data support the potential existence of a specific effect of morphine in the coupling of cannabinoid receptors to GTP-binding proteins, rather than on receptor binding, although this was observed only in the substantia nigra and central gray substance.     

The action of trelibet, a new antidepressive agent on [H]noradrenaline release from rabbit pulmonary artery

Trelibet, a new antidepressant, used at 10⁻⁷–10⁻⁴ M failed to affect the [³H]noradrenaline ([³H]NA) release evoked from the isolated main pulmonary artery of the rabbit low frequency (2 Hz) nerve stimulation whether the neuronal uptake inhibitor cocaine (3 × 10⁻⁵ M) was present or not. Its metabolite (EGYT-2760) however, potentiated the nerve-evoked release of [³H]NA. In the absence of cocaine both the resting and the stimulation-evoked release of ³H increased in response to EGYT-2760. These effect were accompanied by muscle contraction. The EGYT-2760-potentiated transmitter release was inhibited either by exogenously applied 1-noradrenaline (10⁻⁶ M) or clonidine (10⁻⁶ M), preferential agonists of presynaptic ₂-adrenoceptors. The 1-noradrenaline-induced inhibition of transmitter release potentiated by EGYT-2760 was antagonized by 3 × 10⁻⁷ M yohimbine, a preferential ₂-adrenoceptor inhibitor. In the absence of cocaine, Ca²⁺ removal from the external medium failed to affect the ³H outflow-increasing effect of EGYT-2760 but abolished the nerve-evoked release-potentiating action of this compound. It is concluded that the metabolite of trelibet exerts a ‘yohimbine-like’ action, as well as a ‘tyramine-like’ effect in peripheral sympathetic nerve fibres.     

Taurine allosterically inhibits binding of [S]-t-Butylbicyclophosphorothionate (TBPS) to rat brain synaptic membranes

The modulatory effects of taurine on [³⁵S]-t-Butylbicyclophosphorothionate (TBPS) binding to rat brain synaptic membranes were evaluated and compared with that of GABA. Taurine allosterically inhibited TBPS binding by interacting with a bicuculline-sensitive site, similar to GABA. Taurine was as effective as GABA but less potent. The potency of taurine inhibition of TBPS binding varied among brain regions with cerebellum > olfactory bulb > cortex, similar to that of GABA. Inhibition of TBPS binding to cortical membranes measured under nonequilibrium conditions yielded a dynamic biphasic inhibition curve that was similarly shaped for GABA and taurine. The effect of taurine on TBPS binding was pharmacologically specific in that β-alanine and guanadinoethanesulfonate were as effective as taurine, while hypotaurine and -aminoethylhydrogen sulfate were only partially effective at high concentrations, and isethionic acid was without effect. Taurine, similar to GABA enhanced the effects of pentobarbital on TBPS binding when present at concentrations that were otherwise ineffective on their own. The results of these studies support the notion that taurine interacts with the GABA recognition site of the GABA_A receptor complex.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence

γ-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the γ-aminobutyric acid_B Receptor (GABA_BR), but the precise relationship of the GHB receptor (GHBR) to the GABA_BR remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA_BR, we conducted two groups of experiments. First, GABA_BR subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [³H]GHB and [³H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([³H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [³H]GHB and [³H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA_BR1a was deleted. Such mice do not have a functioning GABA_BR. There was no detectable specific [³H]GHB or [³H]NCS-382 binding in HEK 293 cells transfected with GABA_BR1, R2, or R1/R2. Binding to [³H]CGP54626A, a high affinity GABA_BR antagonist, was absent in GABA_BR1a^−/− mice. There was no difference in [³H]NCS-382 binding observed in the brains of GABA_BR1a^−/−, GABA_BR1a^+/− or GABA_BR1a^+/+ mice. Specific [³H]GHB binding was observed in the brain of GABA_BR1a^−/− mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA_BR.     

Thromboxane A(2)-mediated Cl(-) secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

The effects of lanthanum chloride administration in newborn chicks on glutamate uptake and release by brain synaptosomes

Acute i.p. administration of lanthanum chloride to newborn chicks at a single dose of 250 mg/kg body weight causes inhibition of the high affinity uptake of [¹⁴C]glutamate by isolated brain synaptosomes. There is also a marked decrease in the release of preloaded [¹⁴C]glutamate from brain synaptosomes in the presence of externally available Ca²⁺ (1.2 mM) or a high K⁺ concentration (71 mM). The inhibition of glutamate release has been discussed in relation to depletion of Ca²⁺ binding to the synaptosomal membrane under lanthanum intoxication.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

Biphasic effects of mianserin and desipramine on serotonin-evoked current and Cl- efflux in Xenopus oocytes.

Serotonin (5-HT, 1 μM) elicited two phases of Cl⁻ inward current in Xenopus oocytes injected with rat brain mRNA: a transient current (T-current), which was generated rapidly (within 1 min), and a sustained current (S-current), which persisted for 10 min. Each type of 5-HT-evoked response was time-dependent after mRNA injection. The T-current was generated at 20-30 h and the S-current at 30–40 h. Although mianserin at 0.1 μ M completely inhibited the T-current, 10 μ M mianserin was required to suppress the S-current. 5-HT also caused Cl⁻ efflux from oocytes preloaded with ³⁶Cl⁻, Cl⁻ efflux during 1 min, corresponding to the T-current, was inhibited by 0.1 μ M mianserin. A higher concentration of mianserin (10 μ M) was required to block the efflux for 10 min, corresponding to the S-current, as well as the current response. Desipramine selectively inhibited the T-current and Cl⁻ efflux for 1 min. The mechanisms underlying the different sensitivity to mianserin of oocytes injected with rat brain mRNA are discussed.     

Na,K-ATPase activity is selectively increased in thalamus in thiamine deficiency prior to the appearance of neurological symptoms

The relationship between progression of neurological status and the activities of both Na⁺,K⁺- and Mg²⁺-dependent-ATPase (adenosine 5′-triphosphate phosphohydrolase) was investigated in brain regions of pyrithiamine-induced thiamine deficient rats. Thalamic Na⁺,K⁺-ATPase activity was selectively increased by 200% (P < 0.01) prior to the appearance of symptoms of thiamine deficiency and normalized in symptomatic rats. This selective transitory activation precludes a mediation by brain soluble fraction Na⁺,K⁺-ATPase modifiers as does the unaltered distribution in regional high-affinity [³H]ouabain binding densities observed throughout the time-course used in these experiments. Na⁺,K⁺-ATPase maintains cellular ionic gradients and has been implicated in neurotransmitter uptake and release mechanisms. The fact that the increased thalamic Na⁺,K⁺-ATPase activity coincides with the early alterations in serotonin metabolism observed in similarly treated animals and the concomitantly early increase in glucose utilization previously observed in the thalamus of thiamine-deficient rats is discussed.     

Interactions of salsolinol and β-carbolines with cerebral GABA/Benzodiazepine receptor complex: Alteration during alcohol dependence

Effects of salsolinol and 3-carboxy ester of β-carboline, which are known as condensation products of acetaldehyde formed in mammalian brain, on γ-amino-butyric acid (GABA)_A receptor/benzodiazepine receptor/chloride channel complex and its alterations during the development of alcohol dependence were studied. In synaptic membrane preparation, ethyl β-carboline-3-carboxylate (β-ECC) was found to bind to a different site from that for benzodiazepines. The benzodiazepine-stimulated [³H] muscimol binding to GABA_A receptor was suppressed by β-ECC, whereas [³H]β-ECC binding was found to be suppressed by GABA, muscimol and secobarbital. Gel column chromatography using Sephadex G-200 of the solubilized fraction from cerebral particulate fraction by sodium deoxycholate indicated that [³H]β-ECC binding site was eluted in the same fraction (molecular weight = 230,000) as the binding sites for [³H]muscimol, [³H]flunitrazepam, and [³H]t-butylbicycloorthobenzoate, known as a blocker of chloride channel. Salsolinol stimulated the specific bindings of [³H]flunitrazepam and [³H]β-ECC to cerebral synaptic membranes in the presence of chloride anion. GABA-stimulated ³⁶C1-influx into membrane vesicles was stimulated by flunitrazepam, while attenuated by β-ECC, and these effects of flunitrazepam and β-ECC were accentuated by salsolinol. In alcohol dependent mice, the stimulating effect of salsolinol on the bindings of [³H]flunitrazepam and [³H]β-ECC to cerebral synaptic membrane was eliminated. These results indicate that β-ECC binding site may reside on GABA_A receptor complex, and salsolinol may modulate the functions of benzodiazepine and β-ECC binding sites in the brain. The present results also suggest that the alteration of modulatory action of salsolinol on the cerebral [³H]flunitrazepam and [³H]β-ECC binding sites may be involved in the occurrence and/or maintenance of alcohol dependent conditions.     

SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'methyl-4'-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): a novel, potent and selective 5-HT1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT_1B receptors stably expressed in Chinese hamster ovary (CHO) cells (pK_i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT_1D receptors (pK_i 6.6 ± 0.1). Similarly, affinity (pK_i) for rat and guinea pig striatal 5-HT_1B receptors is 9.2 ± 0.1. In [³⁵S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA₂ value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [³⁵S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent pK_B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [³H]-5-HT release from guinea pig and rat cortical slices (S₂/S₁ ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg⁻¹ p.o.) caused a dose-dependent inhibition of ex vivo [³H]-GR125743 binding to rat striatal 5-HT_1B receptors with an ED₅₀ of 2.83 ± 0.39 mg kg⁻¹ p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT_1B autoreceptor antagonist that occupies central 5-HT_1B receptors in vivo following oral administration.     

Effects of L- and N-type Ca channel antagonists on excitatory amino acid-evoked dopamine release

In thc present study we tested the effect of dihydropyridine (DHP) Ca²⁺ channel antagonists and of ω-conotoxin GVIA on [³H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca²⁺ channels in these EAA-mediated processes. Micromolar concentrations (10–30 μM) of DHP L-type Ca²⁺ channel antagonists inhibited [³H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [³H]DA release evoked by the L-type Ca²⁺ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1–1 μM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, ( + )-PN 200-110, was more potent than ( − )-PN 200-110 in inhibiting [³H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca²⁺ channels is able to evoke [³H]DA release. However activation of L-type channels is not involved in EAA-induced [³H]DA release and therefore inhibition of EAA-induccd [³H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca²⁺ channels. ω-Conotoxin GVIA (3 μM) had no effect on [³H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation. ω-Conotoxin GVIA (3 μM) partially inhibited [³H]DA release evoked by NMDA or kainate, suggesting that N-type Ca²⁺ channels could possibly play a role in FAA-mediated responses in these cells.     

Haloperidol competitively inhibits the binding of (+)-[H]SKF-10,047 to σ sites

Scatchard plots of equilibrium saturation binding data revealed that haloperidol inhibits the binding of (+)-[³H]SKF-10,047 to σ sites in a competitive manner. In experiments using membranes from both guinea pig and rat brain the apparent K_d of (+)-[³H]SKF-10,047 for σ sites was significantly increased, whereas the apparent B_max was not altered by the addition of 10 nM haloperidol.